The present invention relates to lysis of cells, and in particular to a system using a laser to induce cell lysis.
Bacterial pathogenesis is one of the most challenging problems worldwide, making it necessary to analyze medical, food and environmental samples for the presence of pathogenic microorganisms. Worldwide, infectious diseases account for approximately 40% of the estimated total 50 million annual deaths. A critical step in detection and identification of pathogenic organisms is very often cell lysis. In order to determine intracellular proteins such as DNA or RNA molecules to identify the organisms, a cell membrane has to be disrupted via a rapid, non-damaging and simple procedure, which ideally does not add adulterating chemicals into the cell lysate or further dilutes the sample. Each sample preparation step in a bioanalytical detection system needs to be designed carefully, especially if single cell detection is targeted. It can be expected that technology for the manipulation and analysis of single cells will play an important role in such areas as biomedical research, drug discover, diagnosis of disease and medical treatment.
Frequently used methods for cell lysis are based on mechanical, physical, chemical or biological principles. In one method, cell walls are disrupted via repeated freeze/thaw cycles, by heating of the cells to temperatures above 60xc2x0 C. or by osmotic pressure. Alternatively, cells are lysed via bead milling, sonication or lyophilization. Biological lysis methods often use the enzyme lysozyme, or bacteriophages, and chemical methods utilize chaotropic agents at high concentrations. Depending on the microorganism, and on the availability of equipment, all of these procedures are currently used in research and industrial laboratories. Main disadvantages of these procedures are labor intensity, time requirement, damage to nucleic acid and proteins and also adulteration of the cell lysate with other compounds, which have to be separated from molecules of interest, such as nucleic acids and proteins.
Laser microsurgery has demonstrated that cell membranes could be destroyed locally while keeping the interior of the cell non-damaged. Wavelengths above 750 nm are typically used on a membrane of a cell that is sucked up in a patch clamp type of device. A shock wave has also been used to lyse cells by placing the cells or cellular components in a solution.
A method of determining laser parameters for lysing cells involves exposing cells from a sub-sample of a sample to laser light. At least one parameter of the laser is varied, and damage to intracellular molecules of sub-samples of the sample at such varied parameters is measured. At least one parameter is determined based on the measured damage. In one embodiment, the laser parameters comprise power, wavelength and duration.
In one embodiment, the sub-sample contains between approximately 80 to 2000 cells and the intracellular molecules comprise RNA. In a further embodiment, measuring damage comprises measuring protein damage.
The wavelength parameter is varied between approximately 500 nm and 3500 nm and higher and the power parameter is varied between approximately 0.0 mW and 300 mW. The duration parameter is approximately 4 minutes but may be varied. For smaller volumes, the duration may be reduced to one second or less.
In a further embodiment, a system for laser lysing cells includes a hydrophobic surface for supporting cell samples, a laser, and a mirror and lens for directing the laser onto the cell samples. The laser has a power level variable between approximately 0.0 mW and 300 mW. The hydrophobic surface comprises an infrared card wrapped in a layer of parafilm.
In a further embodiment, a microchannel system provides a transport mechanism for cells to be lysed. Several parallel channels or a serpentine single channel are two different embodiments. The microchannel system is combined with a laser to lyse cells while they are being transported. The laser is disposed within a trench to expose the cells in the channels in one embodiment. Such a laser does not need lenses, but lenses may be used if desired. In this embodiment, exposure times of one second or less are sufficient to lyse the cells but longer exposure times may be required for different cell types. In still further embodiments, the laser is integrated into a semiconductor substrate in which the channels are formed.